The federally endangered ocelot (Leopardus pardalis) population of south Texas is declining; fewer than an estimated 80 ocelots remain. South Texas has robust transmission of Trypanosoma cruzi, the protozoan parasite causing Chagas disease in humans and various mammals. This parasite's impact in ocelots is unknown. Blood from live-trapped ocelots was collected by US Fish and Wildlife Service personnel in an annual monitoring program; additionally, tissues were obtained from carcasses collected from 2010 to 2017 around Laguna Atascosa National Wildlife Refuge in south Texas and placed in scientific collections. Variable samples were available from 21 ocelots: skeletal muscle (n=15), heart tissue (n=5), lung (n=1), kidney (n=1), spleen (n=1), liver (n=1), blood clot (n=9), and serum (n=3). Overall, 3/21 (14.3%) ocelots showed evidence of T. cruzi infection or exposure, with T. cruzi PCR-positive samples of skeletal muscle, heart, and blood clot, respectively. All three were infected with the T. cruzi discrete taxonomic unit “TcI”; one of these ocelots also had anti–T. cruzi antibodies. Lymphoplasmacytic inflammation was noted in the PCR-positive heart tissue and in some PCR-negative tissues from this and other individuals. Incidentally, Sarcocystis spp. were noted histologically in five ocelots. Trypanosoma cruzi infection and associated cardiac lesions suggest that this parasite should be further investigated in vulnerable populations.